MACARANGIN , A GERANYLATED FLAVONOID AND ANTICANCER ACTIVE COMPOUND ISOLATED FROM ETHYL ACETAT FRACTION OF Macaranga gigantifolia LEAVES

Email: ahmaddarmawan@yahoo.com ABSTRACT Macaranga known locally as mahang-mahangan has uniquely ecological function, and also became a part of traditional medicine in Indonesia. Macaranga genus also known as a sources of terpenoid and phenolic (flavonoid) compounds which have biological activity as antioxidant and anticancer (cytotoxicity).There are few phytochemical investigations have been done on M. gigantifolia species. As a part of our continuing research of isolation anticancer compound from natural product, a geranylated flavonoid compound (macarangin) has been isolated from ethyl acetate fraction of Macaranga gigantifolia leaves using chromatography methods. The isolated compund (isolat MG) was elucidated to gain the chemical structure based on spectroscopic data (LC-MS and FT-NMR). Cytotoxicity test of this compound was tested against MCF 7 cell lines, showed that macarangin has a potential activity with IC50 value 119.12μg/mL.


INTRODUCTION
Macaranga (Euphorbiaceae), known locally as mahang-mahangan has uniquely ecological function, and also became a part of traditional medicine in Indonesia such as for diarrhea, wound and cough (Heyne, 1987).There are more than 308 species of Macaranga, wide-spread from Africa and west region of Madagascar until tropical region of Asia, North Australia and east region of Pacific Islands (Blattner, 2001).Macaranga genus also known as a sources of terpenoid (Hui, 1971)] and phenolic (flavonoid) (Jang, 2002;Schutz, 1995;Sutthivaiyakit, 2002;Yoder, 2007) compounds which have biological activity as antioxidant (Phommart, 2005) and anticancer (cytotoxicity) (Yoder, 2007).Macarangin, a geranylated flavonoid compound is one of the flavonoid typical compounds contained in Macaranga genus, and have been isolated from M. vedeliana (Hnawia, 1990) and M. denticulate (Sutthivaiyakit, 2002).Scopoletin is one of the secondary metabolite compounds was isolated from M. gigantifolia (Darmawan, 2012).There are few phytochemical investigations have been done on M. gigantifolia species.This study reports about the isolation and structure elucidation of macarangin from the ethyl acetate fraction of methanolic extract of M. gigantifolia leaves.This is the first reports about macarangin compound isolated from M. gigantifolia.

MATERIAL AND METHODS
M. gigantifolia leaves collected from Mekongga forest region, Kolaka District, Southeast Sulawesi -Indonesia in March 2013 and identified by Mr. Ismail Rachman from Herbarium Bogoriense, Research Center for Biology-LIPI.

Instrumentation
NMR spectra were recorded on a JEOL JNM-ECA 500 Spectrometer with TMS as an internal standard, and ESI-MS was performed on Mariner Shimadzu ESI system.

Chemical compound characterization
Isolate MG characterized based on spectroscopic data obtained from FT-NMR and LC-MS analysis.FT-NMR and LC-MS analysis performed with JEOL JNM-ECA 500 MHz and Mariner Shimadzu ESI system.

Cytotoxicity test in vitro with Methyl Thyazole Tetrazolium (MTT) Assay
Isolate MG was tested in vitro for its cytotoxicity against MCF-7 cell line.MCF-7 cells growth inhibition by Isolat MG was analyzed 3-(4,5-dimethylthyazole-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay reported by Jin (2010) and Yunianto (2012).MCF-7 cells were seeded in 96 well plates and incubated with MTT (5mg/mL) for 4h.Cells further solubilized by added 100µL of DMSO.Absorbance was read at 570nm.Cell viability in treated cells was expressed as the amount of dye reduction relative compared to untreated control cells.The wells which contained only medium and 10mL of MTT were used as blanks for the plate reader.

Chemical compound characterization
Isolate MG, a yellowish brown paste obtained from two step chromatography process using chromatotron with n-hexane:ethyl acetate as solvent system, gave a molecular ion [M+H] m/z 423.20 in ESI-MS, which mean that [M + ] is m/z 422.20.
From the NMR data ( 1 H-and 13 C-NMR) indicates that Isolat MG is a geranylated flavonoid compound.Data HMBC-NMR showed that methylene group at δ 3.37 (H-1") correlated with three ring A carbon atom C-5, C-6, and C-7, it means geranyl substituent attached to the flavonoid aglycone through A ring (Figure 1.).
Based on NMR data, supported by LC-MS data and reference comparative (Table I.), Isolat MG was identified as macarangin (Figure 2) (Hnawia, 1990).This is the first report about macarangin content in Macaranga gigantifolia.

Cytotoxicity test in vitro with Methyl
Thyazole Tetrazolium (MTT) Assay.II) of Isolat MG was carried out at 100 ppm concentration using MTT viability test against MCF-7 cells (breast cancer cell) showed that Isolat MG could inhibit the growth of MCF-7 cell with IC50 value 119.12μg/mL (Figure 3).It means that Isolat MG has medium potential activity as breast cancer inhibitor.

Table I .
1 H-and 13 C-NMR data of Isolate MG and Macarangin